Project Summary/Abstract There is significant potential to decrease ovarian cancer incidence through prevention. Oral contraceptives (OCs) confer a 30-50% reduced risk of developing epithelial ovarian cancer, suggesting that an effective preventive approach using hormones is possible. Progestin-potent OCs confer twice the ovarian cancer protection as newer weak-progestin OCs. These data suggest that a progestin-based pharmacologic strategy may be highly effective for prevention of ovarian cancer. Progestin potency can be enhanced through combination with vitamin D, a second preventive agent that is also non-toxic when administered as cholecalciferol; therefore, the two agents may act synergistically on chemopreventive endpoints in the gynecologic tract such that progestins positively influence vitamin D metabolism. The tissue of origin of ovarian cancer remains uncertain. There is now compelling evidence that the origin of many high grade ovarian cancers may be the fallopian tube, thus creating an opportunity for identifying molecular targets in the fallopian tube for effective ovarian cancer prevention. The proposed research is designed to determine the molecular mechanisms by which ovarian cancer, whether it originates from the ovary or the fallopian tube, may be prevented. Prevention of fallopian tube/ovarian cancer using a combination of vitamin D and each of the three classes of progestins will be tested in the following three aims. First, the impact of these treatments on molecular pathways that lead to cell death in fallopian tube and ovarian epithelial cells will be investigated. This will include extension of the active life of vitamin D by inhibition of CYP24A1, as well as increased sensitivity of these cells to vitamin D by enhanced production of the vitamin D receptor. Secondly, the interaction between the progesterone receptor and progesterone response elements PRE(s) will be mechanistically defined in the CYP24A1 promoter through the use of CYP24A1 promoter-luciferase reporter constructs. Endogenous functional PRE(s) in ovarian cancer cell lines will be selectively inactivated using CRISPR/Cas9-mediated genome editing. The resulting stable cell line(s) harboring non-functional PRE(s) in the CYP24A1 promoter will then be used to validate loss of synergy between progesterone and vitamin D in vitro as well as in vivo in mouse xenografts. Thirdly, in vivo experiments using two genetically modified mouse strains that develop fallopian tube cancer closely resembling human ovarian cancer will be performed to determine the in vivo impact of progestins and vitamin D on fallopian tube epithelial cancer development. Understanding the mechanisms by which progestins are integrally involved in each of these processes, will facilitate development of a robust pharmacologic approach to prevent ovarian/fallopian tube cancer. The goal of this research is to identify an optimal pharmacologic strategy combining progestins and vitamin D for an effective ovarian cancer chemopreventive approach that can be tested in women at risk of developing ovarian cancer.